Importance of minor histocompatibility antigens in the development of allograft arteriosclerosis.

Although acute rejection has been mostly ameliorated with the use of powerful immunosuppressive drugs, kidney and heart transplants continue to succumb to a more chronic response characterized by intimal lesions in the graft vasculature. This late-stage response is referred to as allograft arteriosclerosis. Allorecognition is clearly involved in the initiation of this response but the relative importance of major histocompatibility (MHC) and minor histocompatibility (mH) antigens remains unclear. By taking advantage of the B10 congenic set of mice and our newly described mouse aortic interposition graft model we have been able to assess the contribution of these antigens to the development of the concentric intimal lesions characteristic of allograft arteriosclerosis. We performed transplants between syngeneic animals, animals which were disparate at both MHC and multiple mH, animals which were disparate at MHC only, and animals which were disparate at multiple mH antigens only. H-Y antigen variation was controlled for by performing all transplants between female mice. In all cases the recipients were C57BL/10 (H-2b) mice. Both cellular infiltration into the intima and resulting intimal thickness were measured at 2, 4, 8, and 13 weeks posttransplant. At all time points, the grafts from MHC disparate only donors showed less severe intimal lesions than the grafts from fully disparate or mH disparate donors. This difference reached statistical significance at 4 and 13 weeks. This suggests that mH antigens are as immunogenic as MHC antigens with respect to the generation of allograft arteriosclerosis. These findings are not unique to vascular grafts and may relate to the importance of indirect antigen presentation in the development of chronic rejection.

Migration of macrophage-like cells within encapsulated islets of Langerhans maintained in tissue culture.

Islets of Langerhans isolated from the pancreas and encapsulated in alginate-polylysine-alginate micro-spheres can potentially serve as a self-regulating supply of insulin in response to glucose loads. A longitudinal ultrastructural and immunohistochemical study of encapsulated rat islets cultured in CMRL-1969 media at a constant glucose concentration of 5.5 mmol/L (100 mg%) allowed several observations. First, acinar cells, which remain attached to isolated islets, disappeared within 1 wk in tissue culture. Damaged endocrine cells also disappeared at this time. Phagocytic cells having ultrastructural features suggesting that they are macrophages emerged from the islets within about a week and ingested portions of the inner layer of capsule polymer. These macrophage-like cells retained these polymers until their death which occurred at around 1-2 mo after isolation; at no time did we observe phagocytic cells actually breaching the microsphere capsules. Beta cells remained well-granulated over 90 days of culture but accumulated lipofuscin-like residual bodies. Under these conditions, these bodies began to accumulate appreciably after about one week in culture.

Morphometric analysis on myocardial injury related to the use of high volume potassium cardioplegic solution during ischemic arrest.

We correlated the effects of high volumes of K+ cardioplegic solution on myocardial structure and function in 16 dogs following open-heart surgery. Eight animals received high volume potassium cardioplegic solution (25 cc/kg body weight, every 30 min) during 90 min of ischemic arrest (HVK-C group). The others received sufficient cardioplegic solution to maintain complete electrical arrest as defined by voltage monitoring criteria (VM group). Cardiac index (CI), left ventricular stroke work index (LVSWI), and myocardial contractility (dp/dt) were determined before arrest and after 90 min of ischemia and 45 min of reperfusion. Biopsies were taken for EM ultrastructure and ATP estimation. Morphometric analysis of EM micrographs found increased volume of damaged mitochondria (DMR) (p less than 0.025), damaged myofibrils (DMF) (p less than 0.001), intermyofibrilar edema (p less than 0.005), T-tubule and sarcoplasmic reticulum (p less than 0.05) in the HVK-C group. Left ventricular (LV) function was more depressed in animals receiving HVK-C. CI decreased by 1.8 +/- 0.4 l/min/square meter (p less than 0.01), LVSWS fell by 3.3 +/- 0.8 gm-m/beat/Kg (p less than 0.01), dp/dt decreased by 684 +/- 135 (p less than 0.0025). ATP decreased by 26% in HVK-C and by 12% in VM group (0.1 less than p less than 0.05). Structural damage (scores of injured volume of mitochondria and myofibrils) correlated with post-ischemic depression of LV function (Cardiac output and myocardial contractility), r = -0.72 and -0.66 (p less than 0.001 and 0.004).

The relationship of mast cells and their secreted products to the volume of fibrosis in posttransplant hearts.

A series of 96 posttransplant endomyocardial biopsies taken from 11 patients was subjected to quantitative analysis of mast cells and fibrosis. Ultrastructural analysis showed that mast cell numbers were increased and there was obvious degranulation in some posttransplant hearts. Activated mast cells and their secreted products, which contain heparin and histamine, are toxic to the hearts and may contribute to interstitial and perimyocytic fibrosis. The numbers of mast cells and granules were correlated with volume of fibrosis (r = 0.63, P less than 0.025; r = 0.73, P less than 0.01). There were differences between the release of mast cell granule contents seen in the posttransplant hearts and the rapid and massive reaction of anaphylactic degranulation of mast cells. Some mast cells progressively lost their dense granule contents, displaying a variety of piecemeal degranulation that indicates a slow degranulation process. These events occurred from the first week; they lasted from weeks to months. The fibrosis developed quickly in the cases with more mast cells and degranulation. The cases with fewer mast cells and granules showed only mild increases in the volume of fibrosis. Mast cells appeared as early as the first posttransplantation week. Patients with greater numbers of mast cells underwent more severe rejection episodes. This study demonstrated that mast cells play an early and important role in the perimyocytic and interstitial fibrosis of posttransplant hearts. Mast cells may also be important in the inflammatory process of rejection reaction. The severity of fibrosis appears related to the density of mast cells and their granules.

Effect of aspirin on intimal proliferation and tissue cholesterol in long-term experimental bypass grafts.

UNLABELLED: A single daily dose of aspirin (ASA) reduces the incidence of early graft thrombosis after coronary bypass operations. Recent data indicate that aspirin may not prevent intimal proliferation and cholesterol uptake in experimental bypass grafts which suggests that aspirin may not improve long-term graft patency. To further clarify the effects of aspirin on intimal proliferation and cholesterol metabolism, we performed femoral interposition vein grafts in 12 dogs receiving a 2% cholesterol diet. Six controls (CON) received the diet alone while the remaining animals received the diet with 160 mg aspirin daily before and for 9 months following operation. A segment of each graft was removed at 3 months for measurement of intimal thickness and tissue cholesterol. The entire graft was then harvested at 9 months. Intimal thickness increased rapidly during the first 3 months. A slow and progressive increase in intimal thickness was observed between 3 and 9 months. There was, however, no difference in intimal thickness between the two groups. Tissue cholesterol increased similarly in both groups. Rapid cholesterol uptake occurred within the first 3 months and then decreased between 3 and 9 months. CONCLUSIONS: (1) ASA failed to reduce intimal proliferation and cholesterol uptake in experimental bypass grafts suggesting that ASA may not prevent late graft failure, (2) Accelerated intimal proliferation and cholesterol uptake occurred within the first 3 months emphasizing the importance of developing and instituting anti-proliferative therapy immediately after aortocoronary bypass.

Myocardial oxygen consumption and lactate production during antegrade warm blood cardioplegia.

UNLABELLED: Continuous warm blood cardioplegia has recently been recommended as an alternative to multidose cold blood cardioplegia for myocardial protection during coronary bypass operations. Cardioplegia may have to be interrupted in order to provide a bloodless operating field during coronary anastomosis. To determine the effects of ischemia at normothermia on myocardial oxygen consumption and lactate production we randomized 17 dogs to receive either warm blood cardioplegia (37 degrees C) or cold blood cardioplegia combined with systemic and topical cooling. After initiating arrest, cardioplegia was interrupted for periods of 1, 2, 3, 4, 5, 6, and 10 min. Myocardial oxygen debt occurred after 3.5 min of ischemia in the 9 animals receiving warm blood cardioplegia. In contrast, myocardial oxygen consumption never exceeded oxygen availability during cold blood cardioplegia (P less than 0.001). Lactate production increased linearly in both groups but was much greater in those animals receiving warm blood cardioplegia (P less than 0.001). Spontaneous electromechanical activity was much more common during warm blood cardioplegia which required frequent infusions of cardioplegia to maintain cardiac arrest (P less than 0.0003). CONCLUSIONS: (1) Oxygen debt occurred after 3.5 min of warm ischemia; (2) spontaneous electromechanical activity is more common during warm heart protection which necessitates the use of larger volumes of cardioplegia to maintain cardiac arrest.

Effect of aspirin on intimal hyperplasia and cholesterol uptake in experimental bypass grafts.

The effects of aspirin on intimal and medial smooth muscle cell proliferation and cholesterol uptake in experimental bypass grafts were examined in a hypercholesterolemic canine model. Ten animals receiving a 2% cholesterol diet served as controls, while a further 10 animals received the same diet and 160 mg aspirin daily. Segments of external jugular vein were implanted between bilaterally divided femoral arteries. Tissue cholesterol and intimal and medial thicknesses were measured at six weeks. Graft cholesterol had increased 1.7 mumol/g at six weeks in the control group but only rose by 0.28 mumol/g in animals receiving aspirin (P less than 0.002). Intimal and medial smooth muscle cell proliferation was evident in all experimental bypass grafts and was unaffected by aspirin. It is concluded that cholesterol uptake and smooth muscle proliferation may be controlled by different mechanisms, and that aspirin reduces cholesterol uptake but does not prevent smooth muscle cell proliferation in experimental bypass grafts.

Oxygenated cardioplegia ameliorates the adverse effects of small amplitude electrical recording of activity on myocardial metabolic and functional recovery.

UNLABELLED: Small amplitude electrical activity has been recorded from the myocardium during cardioplegic arrest in the absence of electromechanical activity. The presence of persistent electrical activity has been associated with impaired myocardial metabolic and functional recovery. To determine whether or not oxygenated cardioplegia would provide sufficient oxygen to support the increased metabolic activity associated with persistent electrical activity during cardioplegic arrest, we randomized 14 adult mongrel dogs to receive either non-oxygenated or oxygenated cardioplegia during 90 min of ischaemia. Cardiac index (CI), left ventricular stroke work index (LVSWI) and dp/dt were measured before bypass and after 90 min of ischaemia and 45 min of reperfusion. Myocardial oxygen consumption (MVO2) and lactate extraction were measured before and after bypass. Intramyocardial voltage was monitored during cardioplegic arrest, and MVO2 was measured during cardioplegia infusion. The onset of small amplitude electrical activity was associated with a rise in intramyocardial voltage and an increase in MVO2. CI, LVSWI and dp/dt were better preserved in those animals receiving oxygenated cardioplegia. MVO2 and lactate consumption following cardioplegia arrest were also higher in this group. CONCLUSIONS: (1) small amplitude electrical activity during cardioplegic arrest is associated with a rise in MVO2. (2) Oxygenated cardioplegia increases myocardial protection by providing oxygen for the increased metabolic activity associated with the presence of this small amplitude electrical activity.

Effect of high-volume cardioplegia on small-amplitude electrical activity during cardioplegia arrest.

UNLABELLED: The effects of high-volume cardioplegia on the presence of small-amplitude electrical activity during cardioplegia arrest were investigated in 19 mongrel dogs. The animals were randomly assigned to receive either high-volume crystalloid cardioplegia (HV-plege) or crystalloid cardioplegia guided by continuous electrical monitoring (V-plege). Cardiac index, left ventricular stroke work index dp/dt, and myocardial oxygen consumption were measured before bypass and following 90 min ischemia and 45 min reperfusion. Biopsies were taken for measurement of adenosine triphosphate (ATP) and examination of myocardial ultrastructure. Nine animals received HV-plege, while the remaining 10 animals received cardioplegia guided by voltage criteria. Small-amplitude electrical potentials were recorded within 10-15 min after the infusion of cardioplegia in all animals receiving cardioplegia guided by voltage criteria. Electrical activity, however, was immediately abolished by reinfusion of cardioplegia. HV-plege reduced the incidence of small-amplitude electrical activity during cardioplegia arrest but did not prevent electrical activity. Left ventricular function and myocardial ultrastructure were better preserved when cardioplegia was guided by electrical monitoring. ATP decreased similarly in both groups following cardioplegic arrest, but myocardial oxygen consumption was significantly higher following the arrest in the V-plege group. CONCLUSIONS: HV-plege does not prevent small-amplitude electrical activity and may have adverse effects on myocardial metabolic and functional recovery.

The effects of low, medium and high dose aspirin on intimal proliferation in autologous vein grafts used for arterial reconstruction.

Bilateral femoral vein grafts were implanted in 47 adult mongrel dogs to determine the effects of aspirin on intimal hyperplasia. The animals were fed a commercially-prepared 2% cholesterol diet before and for 6 weeks following operation. Twelve animals served as the controls while the remaining animals were divided into three groups receiving low, medium, and high dose aspirin. Eleven animals received 75 mg of aspirin daily, 11 animals were fed 225 mg of aspirin daily and the remaining 13 animals received 650 mg of aspirin daily. A coagulation profile was carried out before operation and at 2, 4 and 6 weeks following operation. The grafts were harvested at 6 weeks and intimal thickness was measured with a Zeiss computerized interactive image-analyzing system. The prothrombin time, partial thromboplastin time, and platelet count were unchanged in all animals. The bleeding time was prolonged in animals receiving aspirin (P less than 0.02). Intimal thickness measured 4 +/- 0.2 microns before implantation and increased at 6 weeks to 39 +/- 5 microns in the control group. Aspirin failed to reduce intimal hyperplasia. Intimal thickness measured 37 +/- 2 microns in those animals receiving 75 mg of aspirin daily, 35 +/- 3 microns after a daily dose of 225 mg of aspirin and 51 +/- 4 microns in the high dose group receiving 650 mg of aspirin daily. Our data indicates that aspirin fails to reduce intimal proliferation in canine vein grafts which suggests that alternative or combined drug therapy may be necessary to reduce the incidence of late graft failure.

Effects of low-dose marine oils on intimal hyperplasia in autologous vein grafts.

The effects of low-dose cod-liver oil on intimal hyperplasia of vein grafts were examined in 45 adult mongrel dogs undergoing peripheral arterial reconstruction. Fifteen animals served as the control group, 15 animals were fed a fish-oil supplement containing 240 mg of eicosapentaenoic acid daily, and a further 15 animals received 480 mg of eicosapentaenoic acid daily. Segments of undistended external jugular vein were anastomosed to bilaterally divided femoral arteries. The grafts were harvested at 6 weeks and intimal thickness was measured with a computerized interactive image analyzing system. Serum cholesterol level, prothrombin time, partial thromboplastin time, bleeding time, and platelet counts were measured before the operation and at 2, 4, and 6 weeks after the operation. Plasma levels of thromboxane B2 and prostaglandin F1 alpha and serum levels of eicosapentaenoic acid were measured before and 4 weeks after the operation. Serum cholesterol level increased similarly and significantly in all animals. Serum levels of eicosapentaenoic acid rose proportionately with the oral ingestion of fish oil but did not affect coagulation parameters. Plasma thromboxane B2 and prostaglandin F1 alpha were not significantly affected by the ingestion of marine oils. Intimal thickness was 39 +/- 5 microns in the control dogs. Ingestion of 240 mg of eicosapentaenoic acid reduced intimal thickness to 24 +/- 3 microns at 6 weeks (p less than 0.01). Increasing the dose by a factor of 2 did not decrease intimal thickness further, the intima being 23 +/- 2 microns (p less than 0.005). Our data indicate that small doses of fish oil will reduce intimal proliferation in autologous vein grafts and that marine oils may exert their beneficial effects on intimal hyperplasia by a mechanism other than their known effects on prostanoid metabolism.

Comparison of serum sodium and chloride results for Flame IV-Auto Analyzer II, SMAC, Ektachem 400, and Nova 4 clinical analyzer systems.

We determined serum Na+ and Cl- results using Technicon's Flame IV-Auto Analyzer II (FLIV/AAII) system and Kodak's Ektachem 400 clinical analyzer. Our objective was to determine whether Na+ and Cl- results from these analyzers were sufficiently similar to report to clinicians without reference to the system used for the determination. Method precision of the two systems for Na+ results was comparable; whereas Ektachem 400 Cl- results were more imprecise than those determined using the FLIV/AAII, Ektachem Na+ results showed lower correlation with the FLIV/AAII (r = 0.890) and Cl- results were more highly correlated (r = 0.960). When Kodak's newly developed equi-transferant electrolyte reference fluid (ETRF) was used with generation 4 Na+ slides and generation 1 Cl- slides the largest difference observed was 7.0 mmol/L for both Na+ and Cl- results. Using Kodak calibrators and the manufacturer's operational conditions for the Ektachem 400, we observed that a considerable number of sample results for both Na+ and Cl- did not agree within 3.0 mmol/L of the FLIV/AAII values. To corroborate our finding, we also analyzed serum Na+ and Cl- using a Technicon Sequential Multiple Analyzer + Computer (SMAC) system and a Nova 4 + 4 Clinical Analyzer (Nova). We conclude that flame emission systems and direct ion specific electrode systems do not yield comparable Na+ and Cl- results even when total protein and triglyceride concentrations of the samples are within reference ranges.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between the effects of aspirin and dipyridamole on platelet function and prevention of intimal hyperplasia in autologous vein grafts.

Fifty-four adult mongrel dogs receiving a lipid-supplemented diet were used to determine the effects of aspirin and dipyridamole on vein graft intimal hyperplasia. Twenty-one animals received the diet alone, 17 animals received a combination of dipyridamole and aspirin, while a further 16 animals received dipyridamole. Segments of undistended external jugular vein were anastomosed to bilaterally-divided femoral arteries. The vein grafts were harvested at six weeks and intimal thickness was measured with a Zeiss computerized microscope. Serum cholesterol, prothrombin time, partial thromboplastin time and clotting time was measured before the diet and at two, four and six weeks after operation. Plasma thromboxane B2 (TXB2) and the metabolite of prostacyclin I2 (6-keto PGF1 alpha) were determined by radioimmunoassay before and four weeks following operation. A similar and significant increase in serum cholesterol was observed in all animals receiving lipid supplementation. Platelet counts were significantly decreased in those animals receiving a combination of aspirin and dipyridamole while all other hematological parameters remained unchanged. Plasma TXB2 and 6-keto PGF1 alpha were unaffected by dipyridamole but were significantly decreased in those animals receiving the combined drug regimen. Intimal thickness measured 59 +/- 6 micron at six weeks in the controls. Dipyridamole reduced intimal thickness to 26 +/- 2 micron while aspirin and dipyridamole decreased intimal thickness to 28 +/- 2 micron. The data indicate that dipyridamole was as effective in reducing smooth muscle cell proliferation as the combination of aspirin and dipyridamole. Furthermore, the data suggest that antiplatelet drug regimens may reduce intimal thickening in autologous vein grafts by a mechanism other than affecting the thromboxane:prostacyclin ratio.

Prevention of myocardial electrical activity during ischemic arrest with verapamil cardioplegia.

The effect of potassium cardioplegia and potassium cardioplegia containing verapamil hydrochloride on myocardial preservation and electrical activity during prolonged aortic occlusion was examined in 40 adult mongrel dogs. Twenty-four animals (Group 1) received potassium cardioplegia, and 16 animals (Group 2) received potassium verapamil cardioplegia. Potassium or potassium verapamil cardioplegia, 10 ml per kilogram of body weight, was administered after application of the aortic cross-clamp and at 30-minute intervals during the 90-minute arrest. Myocardial temperature was maintained within a range of 8 degrees to 10 degrees C with topical ice saline solution, and electrical activity was monitored with specially designed plunge electrodes. Plunge electrode activity was recorded from the myocardium during arrest in 16 of the 24 animals in Group 1; no electrical activity was present in the animals in Group 2 (p less than .001). The addition of verapamil to potassium cardioplegia increased the tolerance of the myocardium to prolonged ischemia and resulted in less depletion of high-energy phosphate stores and better preservation of mitochondrial ultrastructure and left ventricular function. These data suggest that verapamil augments the preservation provided by potassium cardioplegia by initiating and maintaining a more complete electrical arrest.

Effects of cod-liver oil on intimal hyperplasia in vein grafts used for arterial bypass.

Cod-liver oil rich in eicosapentaenoic acid, an unsaturated fatty acid, has been shown to inhibit platelet aggregation. To determine the effect of this acid on vein-graft intimal hyperplasia, 46 segments of undistended external jugular vein were interposed between the bilaterally divided femoral arteries of 26 mongrel dogs. The animals received a 2% cholesterol diet for 1 week before and 6 weeks after the operation. Eight control animals received the diet alone, eight received cod-liver oil containing 1.8 g of eicosapentaenoic acid daily, for 1 week before and 6 weeks after operation, and seven animals received 1.8 g of eicosapentaenoic acid daily for 6 weeks after operation. Intimal thickness was measured at 6 weeks with a Zeiss computerized interactive image analysing system from multiple cross-sections of vein graft; 395 +/- 10 measurements were made from each graft. The intima measured 4 +/- 0.2 micron (SEM) before implantation and increased to 83 +/- 10 micron in the controls. Eicosapentaenoic acid administered before and after operation reduced intimal hyperplasia to 24 +/- 2.5 micron (p less than 0.001) and to 30 +/- 5 micron in animals receiving eicosapentaenoic acid after operation only (p less than 0.001). These results indicate that the acid inhibits intimal hyperplasia of canine vein grafts but that it is more effective when given before operation (p less than 0.01).

Acinar structure and function in canine pancreatic autografts with duct drainage into the urinary bladder.

Autotransplants of pancreas in 8 dogs, with exocrine drainage into the urinary bladder, were stimulated in vivo with cholecystokinin-pancreozymin (CCK-PZ). Transplant biopsies, when compared with 6 normal pancreases, showed normal acinar structure by light and electron microscopy 13-18 months after initial surgery; 2 transplants with sutures unintentionally transecting ducts were fibrosed and had duct obstruction. After in vivo stimulation, the normal-appearing transplants produced a 7-fold increase in urinary amylase, and quantitative electron microscopy showed a 50% reduction in mature zymogen granules; there were no intracellular organelle abnormalities prior or subsequent to stimulation. Fibrosed transplants produced lesser urinary amylase both prior to and after stimulation. In vitro stimulation of grafts with normal structure increased amylase secretion from 1.5-2.1-fold. In vitro dose-response showed a maximum at 10(-9)M cholecystokinin-octopeptide (CCK-OP) in transplant and control. The in vivo stimulation is more responsive and may be useful for clinical monitoring of graft survival. In vivo stimulation occurred after induced urinary tract infection; because no pancreatitis ensued, a regimen of trophic stimulation by CCK-PZ was not contraindicated. The bladder tolerated exocrine drainage with no significant change, and bladder infection did not adversely affect the transplant. The islets appeared normal in the transplants by light and qualitative electron microscopic observation; fasting blood glucose and insulin values were normal during the 12-18-month follow-up. Bladder drainage of segmental grafts of pancreas provides a preparation with intact acinar-islet relationships; the present observations suggest that this may permit longer islet survival in the absence of acinar destruction and subsequent fibrosis.

Continuous-flow enzymatic determination of high-density lipoprotein cholesterol.

A simple, sensitive, and precise continuous-flow (Technicon Auto Analyser II) method using enzymic reagents to measure high-density lipoprotein (HDL) cholesterol in the supernatant of serum treated with heparin-manganese reagent is described. 1. The sensitivity of the system was improved by increasing the flow rates of both sample and re-sample manifold lines to 0.23 ml/min. This allowed a recorder full-scale deflection equivalent to 840 mg cholesterol per litre, and permitted a precise determination of cholesterol in the 200-600 mg/l range. 2. Interaction between Mn++ and the enyzmic reagents caused false elevations of the peak plateau on the chart readings, especially when 2M MnCl2 was used. Addition of disodium ethylene-diaminetetra acetate (8 mmol/L) to the enzyme reagent eliminated the spike-like interference in the recorder tracings, and permitted more accurate chart readings. 3. The heparin-Mn++ reagents contributed to the measured HDL-cholesterol. Use of a saline blank improved precision. 4. The inter-assay variation was determined by analysing (n = 26) 2 quality control serum pools: X1 = 362 +/- 11.5 mg/l (CV = 3.18%) and X2 = 252 +/- 14.4 mg/l (CV = 5.74%).

Continuous-flow enzymatic determination of total serum cholesterol and method standardization with CDC-calibrated pooled sera.

We compared Boehringer Mannheim's enzymatic kit for the continuous-flow (AutoAnalyzer II) determination of serum cholesterol with Technicon's N-24a extraction method. Results for patients' samples analyzed by the enzymatic method were higher than those by the comparison method. To evaluate accuracy in the cholesterol determinations, we enrolled the enzymatic method into the Center for Disease Control's (CDC) Lipid Standardization program. We calibrated the method by use of a pooled sera for which cholesterol content was assigned by CDC after analysis by their reference Abell-Kendall procedure. We discuss the difficulties with available calibration material and limitations in the application of some commercial control materials to the enzymatic cholesterol method. The continuous-flow variables, Michaelis-Menton constants, percent ester-hydrolase activity, and other factors affecting the performance of the enzyme-linked cholesterol method are evaluated. We believe pooled sera with an assigned value for cholesterol content is the best calibrator material.